FADS1-3 gene cluster. Among 390 infants of the Indonesian ISADI study, GPL-FA were measured in umbilical plasma (P-0y) and in buccal cells at birth (B-0y), and again in buccal cells at AD onset or one year (B-1y). Prospective and cross-sectional associations with AD were assessed by logistic regression. Interactions of GPL-FA with 14 SNP were tested assuming an additive model. AD was diagnosed in 15.4% of participants. In B-1y, C18:2n-6 was inversely associated with AD; and positive associations were observed for C18:1n-9, C20:4n-6, C22:6n-3 and C20:4n-6/C18:2n-6. There were no prospective associations with AD, however, a significant interaction between the SNP rs174449 and B-0y C14:0 (myristic acid) was observed. This study indicates that Indonesian infants with AD have increased rates of endogenous long-chain polyunsaturated fatty acid production, as well as higher C18:1n-9 levels. GPL-FA measured at birth do not predict later AD incidence; however, genotype interactions reveal novel effects of myristic acid, which are modified by a FADS3 variant.Fatty acids exert a range of different biological activities that could be relevant in the development of atopic dermatitis (AD). This study investigated the association of glycerophospholipid fatty acids (GPL-FA) with AD, and their interactions with single nucleotide polymorphisms (SNP) of the  Atopic dermatitis (AD) is one of the most common inflammatory diseases among infants, with about 60% of cases manifesting in the first year of life . The disFADS genes) [FADS gene variants can modulate the association between dietary fatty acids and allergic diseases [Fatty acids are involved in a range of different biological activities, including intracellular signaling pathways, gene expression, and the production of bioactive lipid mediators . As consS genes) . Gene–didiseases ,20.An important finding among Indonesian participants of the ISADI (Indonesian Prospective Study of Atopic Dermatitis in Infants) birth cohort, was that in contrast to Europeans, alleles predicting a fast conversion of dietary PUFA to LC-PUFA are only carried by a minority of the population . Thus, oFADS gene variants in relation to AD incidence in a South East Asian population. The ISADI study was specifically designed with this intention, comprising a large sample of infants with several genotyped variants of the FADS1-3 gene cluster, and fatty acids measured in buccal cheek cells collected at birth and again at the time of AD diagnosis or at 1 year of age. Since blood collection is not always readily accepted among infants or their parents, the analysis of fatty acids in buccal cells offered a valuable alternative within the present study setting [To our knowledge, no study has yet assessed infant fatty acids and their interactions with  setting .FADS1-3 gene polymorphisms and LC-PUFA composition on the incidence of atopic dermatitis. The study protocol has been described in detail elsewhere [The study involved 390 infants enrolled in the ISADI study, conducted at the Kemayoran Primary Health Care, central Jakarta, from April 2014 to December 2015. The primary endpoint of the ISADI study is to assess the role of lsewhere . Brieflyclinicaltrials.gov as NCT02401178.Physical examinations were performed every 3 months to assess AD, diagnosed by Hanifin-Rajka criteria. Information on relevant covariates was collected by questionnaires. From birth up to 12 months, monthly monitoring by telephone was carried out to record breastfeeding and complementary feeding practices. The present study was conducted according to the International Ethical Guidelines for Biomedical Research Involving Human Subjects. All procedures were approved by the Permanent Medical Research Ethics Committee in Medicine and Health/Faculty of Medicine Universitas Indonesia/Dr Cipto Mangunkusumo Hospital . Written informed consent was obtained from the parents of all study subjects. The study was registered at g for 10 min at 4 °C and the supernatant removed. The same procedure was followed for the second sample of buccal cells. All specimens were immediately frozen at −80 °C and transported on dry ice by air to LMU Munich, Germany, for the analysis of GPL-FA.Umbilical artery plasma (P-0y) and buccal cheek cells (B-0y) were sampled at birth. A second buccal cheek specimen was obtained at AD diagnosis or at age 1 year (B-1y). Umbilical artery plasma was collected into EDTA tubes directly after birth before cord clamping. Specimens were immediately centrifuged. The plasma and buffy coat were frozen at −80 °C. Buccal cheek cells were collected within 1 h of birth by brushing the surfaces of the inner mouth mucosa 20–25 times with gentle pressure, using a Gynobrush . The brush was then put into a Sarstedt tube  and held in place by the tube cap, so that the cells stuck to the brush made sedimentation after centrifugation, as described in patent EUPCT/EP2011/003829. Centrifugation was performed at 1400× g; 10 min; 4 °C) and the supernatant transferred to another vial. Sodium methoxide solution was added for fatty acid methyl ester (FAME) synthesis from GPL-FA at room temperature. After 4 min, the reaction was stopped with methanolic HCl, and the FAME was extracted into 1 mL hexane for gas chromatographic analysis. The FAME was quantified by gas chromatography with flame ionization detection , using a 50-m, 0.22-mm inner diameter BPX70 column . Injection was performed with a programmable temperature vaporizer  to avoid preconcentration of hexane extracts before gas chromatography. FAME peaks were identified and calibrated relative to pentadecanoic acid methyl ester  by comparison with a FAME standard mixture . A total of 27 GPL-FA were measured and are reported as a percentage of total GPL-FA (wt%) [Umbilical plasma and buccal cheek cell GPL-FA were quantified as described previously ,23. BrieGenotyping was performed at the Research Unit of Molecular Epidemiology at Helmholtz Zentrum Munich, Germany, as previously described . DNA wasAll statistical analyses were performed using the statistical software R, version 3.6.2 . Descripρ, i.e., between (a) P-0y and B-0y, (b) P-0y and B-1y, and (c) B-0y and B-1y. Associations between GPL-FA and AD were tested by logistic regression, adjusting for selected covariates . Allele frequency, Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium were assessed among the 18 tested SNP, using the R-package Genetics [FADS variants, interactions between each GPL-FA and each SNP were tested. In the case of a significant interaction, additional analyses were performed for the corresponding GPL-FA, stratified by the relevant SNP genotype. Statistical significance was defined using Bonferroni correction for multiple testing, dividing 0.05 by the number of GPL-FA analyzed . When assessing the interaction effects, the alpha level was further divided by 11, which is the number of effective loci among the analyzed SNP computed according to Nyholt [https://CRAN.R-project.org/package=poolr, accessed on 13 October 2022). Results are presented as odds ratio (OR) and 95% confidence interval (95%CI) for an interquartile range increase in the respective GPL-FA being tested.Outliers in the levels of GPL-FA measured in P-0y, B-0y and B-1y were visually identified by means of boxplots, and these values were removed prior to further analyses. Levels of GPL-FA in each sample, and the relevant ratios thereof, were described by medians (interquartile range). Correlations between GPL-FA levels in paired samples were tested by Spearman’s Genetics . Genotypo Nyholt , using to Nyholt  , and data on SNP genotype, AD and all selected covariates were collected. Descriptive characteristics for the total study population, subjects with AD, and subjects without AD, are summarized in Subjects with AD comprised 15.4 % of the population. These had a higher parental education compared to subjects without AD (15% vs. 7%), were more likely to have a family history of atopy (77% vs. 35%), and were more frequently sick (median of 24 days vs. 9 days).The levels of GPL-FA (wt%) measured in each sample are shown in Correlations between GPL-FAs measured in different samples can be found in the p = 0.0015); and a positive association was observed with C18:1n-9 , C20:4n-6 , C22:6n-3 , and ARA/LA .Results of logistic regression analyses are presented in p-values of the 18 selected SNPs are presented in The minor allele frequencies (MAF) and HWE p = 0.00017). In subsequent analyses stratified by genotype, we observed nominally significant associations in both homozygous major and homozygous minor allele carriers (p < 0.05). Among those who were homozygous major (C/C), higher myristic acid levels were associated with reduced odds of AD incidence , whereas among those who were homozygous minor (T/T), higher odds of AD were observed . Heterozygous allele carriers presented no association of myristic acid with AD   .FADS1-3 gene cluster in this population.We analyzed the association of different fatty acids with the onset of AD up to one year of age in a large Indonesian birth cohort, testing both prospective and cross-sectional associations. Furthermore, we were interested in understanding the interaction of different fatty acids with variants of the The incidence of AD from birth to one year was 15.4%, slightly above the 12-month prevalence of 10.9–12.9% reported in a recent multinational epidemiologic study among children aged 6 months to 6 years in East Asian populations . These vρ > 0.5). Interestingly, the ARA/DGLA ratio reflecting Δ5 desaturase activity was strongly positively correlated; however, the GLA/LA and DHA/EPA ratios reflecting Δ6 desaturase activity were not. Correlations between the GPL-FA measured at birth and levels measured in buccal cheek cells at AD onset or at 1 year were weak, indicating a highly variable fatty acid profile within this brief period. Descriptive statistics demonstrated that the DHA/EPA ratio was much higher in P-0y than in B-0y and B-1y , whereas EPA levels were similar across samples, thus possibly reflecting preferential materno-fetal DHA transfer across the placenta in P-0y [Correlations between fatty acids in different samples were the strongest between umbilical plasma and buccal cell samples collected at birth (P-0y and B-0y). LA and DHA presented the strongest positive correlations between their respective levels in the two samples  that this 4,14-sphingadiene base was identified in human skin for the first time [cis double bond is suggested to decrease the packing density of the lipid bilayer and weaken the lipid–lipid interaction [Our study demonstrates a significant interaction between rs174449 and myristic acid (C14:0) measured in buccal cells at birth (B-0y), which, to our knowledge, has not been reported previously. Stratified analyses indicated a positive association of myristic acid with AD in homozygous carriers of the minor allele (T/T), while major allele carriers (C/C) demonstrated an inverse association. Other correlated SNP presented at least nominally significant interactions with myristic acid (data not shown), suggesting that the observed interaction is unlikely a chance finding. Evidence indicating a role of myristic acid in the risk of AD is so far lacking. Myristic acid can be obtained from the diet, from sources such as dairy (constituting 8–14% of bovine milk ), but itsaturase . Specifirst time . The beneraction . While teraction ,56. Our eraction .A major strength of the present study is the inclusion of a sizeable number of infants with a comprehensive assessment of the GPL-FA profile and doctor-diagnosed AD. We measured GPL-FA in buccal cheek cells, allowing for a less invasive sampling procedure and likely increased participation levels as a result. The further inclusion of umbilical artery plasma (P-0y) allowed for the comparison of the fatty acid levels across samples, while also highlighting clear sample-specific differences in association with AD. Furthermore, the study did not include participants whose mothers took omega-3 or omega-6 supplements, which would potentially dilute the effects resulting from the natural enzymatic pathway. To our knowledge, this is the first study in an Indonesian population to investigate the interaction of genetic variation, nutrition, and the incidence of AD in infants. Nevertheless, given the observational nature of the present study, we cannot infer causal effects, even though the prospective analyses are less likely to be affected by reverse causation.FADS3 gene variant rs174449, and might offer new opportunities for precision prevention strategies.We conclude that Indonesian infants developing AD within the first year of life have higher rates of Δ5 fatty acid desaturase activity, as reflected by higher ARA and lower LA levels as well as a higher ARA/LA ratio. Oleic acid levels are also positively associated with AD at the time of disease diagnosis. Fatty acid levels measured in cord blood or buccal cells at birth are not associated with AD incidence. However, genotype interaction analyses indicate an effect of myristic acid levels measured at birth on AD incidence, which is modified by the


Objective: The purpose of this study was to establish an N6-methylandenosine (m6A)-related long non-coding RNA (lncRNA) signature to predict the prognosis of hepatocellular carcinoma (HCC).Methods: Pearson correlation analysis was used to identify m6A-related lncRNAs. We then performed univariate Cox regression analysis and least absolute shrinkage and selection operator (LASSO) Cox regression analysis to construct an m6A-related lncRNA signature. Based on the cutoff value of the risk score determined by the X-title software, we divided the HCC patients into high -and low-risk groups. A time-dependent ROC curve was used to evaluate the predictive value of the model. Finally, we constructed a nomogram based on the m6A-related lncRNA signature.Results: ZEB1-AS1, MIR210HG, BACE1-AS, and SNHG3 were identified to comprise an m6A-related lncRNA signature. These four lncRNAs were upregulated in HCC tissues compared to normal tissues. The prognosis of patients with HCC in the low-risk group was significantly longer than that in the high-risk group. The M6A-related lncRNA signature was significantly associated with clinicopathological features and was established as a risk factor for the prognosis of patients with HCC. The nomogram based on the m6A-related lncRNA signature had a good distinguishing ability and consistency.Conclusion: We identified an m6A-related lncRNA signature and constructed a nomogram model to evaluate the prognosis of patients with HCC. Hepatocellular carcinoma (HCC), the primary common type of liver cancer, ranks third among cancer-related deaths worldwide . DespiteSince Desrosiers first described N6-methyladenosine (m6A) in 1974, researchers have discovered that it is a widely present modification of non-coding RNAs and mRNAs in eukaryotes . Furtherhttps://portal.gdc.cancer.gov). ICGC RNA sequencing data and corresponding clinicopathological data were downloaded from the corresponding official website (https://dcc.icgc.org/projects/LIRI-JP). The HCC cohorts of TCGA and ICGC datasets contained RNA sequencing data of 370 and 232 HCC patients, respectively. Considering that HCC patients with a survival time of less than 30 days are likely to die due to non-tumor factors, we included 342 HCC patients in the TCGA cohort and 230 HCC patients in the ICGC cohort for subsequent analysis. According to previously published research of Tu et al., we identified 21 m6A regulatory factors, including readers , erasers (ALKBH5 and FTO), and writers  (We downloaded RNA-seq data (FPKM) and corresponding clinicopathological characteristics of HCC patients from the TCGA official website ( METTL3) . The exphttps://www.gencodegenes.org/) and annotated the ensemble IDs in the TCGA and ICGC datasets. Finally, we annotated 14,091 lncRNAs in the TCGA HCC dataset and 257 lncRNAs in the ICGC HCC dataset. The Perl language was used to extract the lncRNA expression matrix from TCGA and ICGC HCC datasets. Based on Wu’s research, we defined m6A-related lncRNAs as those with an absolute value of correlation coefficient between lncRNA expression and m6A regulators higher than 0.3, and a p-value less than 0.001  according to the manufacturer’s instructions. The PCR-specific primers were determined using GENERAL BIOL  and used according to the manufacturer’s instructions. RNA was converted to cDNA using the PrimeScript RT Polymerase (Takara) reverse transcription kit to determine lncRNA mRNA expression. The fold change was calculated using the 2To explore whether m6A-related lncRNA risk score is an independent factor predicting the prognosis of patients with HCC, we also included clinical data such as age, sex, TNM stage, and histological grade into the Cox regression model. Univariate and multivariate Cox regression analyses were performed sequentially to screen for independent risk factors that affected the prognosis of patients with HCC. We constructed a nomogram based on variables selected using multivariate Cox regression analysis. The nomogram’s distinguishing ability was determined using the C-index and the area under the time-dependent ROC curve, and its consistency is shown using the 1-year, 2-year, and 3-year calibration curves.http://software.broadinstitute.org/Gsea/downloads.jsp) was used to explore the potential pathways for the role of m6A-related lncRNAs in HCC. Based on the selected cutoff value for the TCGA dataset, the TCGA HCC cohort was divided into low- and high-risk groups. Therefore, an annotation file (.cls) required by GSEA was obtained. Next, we imported the expression profile containing 56,753 genes and 342 HCC samples into the Morpheus website (https://software.broadinstitute.org/morpheus/) to obtain the expression matrix (.gct), as determined by the GSEA. Finally, we uploaded these two files to GSEA software and set the gene set to c2.cp.kegg.v7.2.symbols.gmt. The permutation parameter was set to 1,000. A p-value less than 0.05 and FDR less than 0.25 were considered to indicate statistically significant differences.The Gene set enrichment analysis (GSEA) (https://cistrome.shinyapps.io/timer/) contains six immune cell components in the TCGA dataset, including CD8 T cells, neutrophils, CD4 T cells, B cells, macrophages, and dendritic cells. Based on the value of the m6A-related lncRNA risk score constructed in the TCGA dataset, we performed Spearman’s correlation analysis to explore the correlation between the risk score and the level of immune cell infiltration.The timer database  to describe the variables that met the normal distribution and used the median (interquartile range) (IQR) to describe the variables that did not meet the normal distribution. Pearson correlation analysis was used to analyze the correlation between m6A regulators and lncRNAs in TCGA and ICGC HCC datasets. Statistical analysis was performed using R software (version 4.0.3.) and the SPSS software (version 25.0). A double-sided After excluding patients with HCC with missing survival time or survival time of less than 30 days, we finally included 342 HCC patients in the TCGA cohort and 230 HCC patients in the ICGC cohort for follow-up analysis. Detailed information on the patients with HCC included in this study is presented in p <0.05) (p <0.0001)  . We thenp <0.05) . Finallyp <0.05) . These fp <0.05) . The heap <0.05) . Accordip <0.05) . Therefo<0.0001) . Moreove<0.0001) . Hence, p = 0.017)  . The res= 0.017) . This pop = 0.023), histological grade (p <0.001), TNM stage (p = 0.002), and survival status (p <0.001). In the ICGC dataset, we found that the m6A-related lncRNA signature was significantly associated with sex (p = 0.012), TNM stage (p = 0.022), and survival status (p = 0.004), which is consistent with the results of TCGA (p = 0.004)  were analyzed. The m6A-related lncRNA signature was significantly associated with sex ( of TCGA . In the = 0.004) .p <0.001) and risk score (p <0.001) were significantly related to the overall survival of patients with HCC. We then input these two variables into the multivariate Cox regression analysis, and the results showed that the m6A-related lncRNA risk score was an independent risk factor for the overall survival of patients with HCC (p <0.001) (p = 0.025) (p <0.001), cirrhosis (p = 0.032), and risk score (p = 0.022) were significantly associated with OS. Multivariate Cox regression analysis showed that TNM stage (p <0.001) was an independent risk factor for OS in patients with HCC  . Similar= 0.025) . Howeverwith HCC .To intuitively assess the prognosis of patients with HCC, we established a predictive nomogram model. According to the multivariate Cox regression analysis results, we included the m6A-related lncRNA risk scores and TNM stages to construct a nomogram model . In the To further identify the molecular mechanism of the m6A-related lncRNA signature in patients with HCC, we divided 342 HCC patients in the TCGA dataset into high- and low-risk groups based on the previous cutoff value (2.83). The enrichment pathways of the high-risk group were mainly enriched in complement and coagulation cascades, drug metabolism cytochrome P450, retinol metabolism, primary bile acid biosynthesis, and fatty acid metabolism (Top 5). The enrichment pathways of the high-risk group were mainly concentrated in base excision repair, spliceosome, pyrimidine metabolism, RNA degradation, and nucleotide excision repair (Top 5) . The detr = 0.150; p = 0.005), CD4 T cells , B cells , macrophages , and dendritic cells   . This shWith the deepening of lncRNAs research in recent years, their correlation with m6A regulators has gradually attracted the attention of scientific researchers. There has been a lot of published literature about m6A regulators or lncRNA signature prediction models used to predict the prognosis of patients with tumors, especially HCC . For exaOPLS-DA is a supervised statistical discriminant analysis (LDA) method. This method uses partial least squares regression to establish a relationship between lncRNA expression and dependent variables. It uses orthogonal signal correction technology to decompose the X matrix information into two types of information related to Y and unrelated to Y, and then filters out the information irrelevant to the classification. The relevant information is mainly concentrated in the first prediction component. It can effectively reduce the complexity of the model and enhance its explanatory power without reducing its predictive ability. It is generally believed that in binary classification questions, OPLS-DA can be used as the preferred analysis method, which is often used in metabolomic studies . Robert In our study, through the integration of TCGA and ICGC datasets, we determined an m6A-related lncRNA signature for predicting the prognosis of patients with HCC. Based on the cutoff value of the m6A-related lncRNA risk score for each dataset, we divided the patients with HCC into low-and high-risk groups. The overall survival rate of patients with HCC in the high-risk group was significantly lower than that in the low-risk group. In the TCGA training dataset, the results of multivariate Cox regression analysis showed that the risk score and tumor stage were independent risk factors for the prognosis of patients with HCC; therefore, we constructed a nomogram model based on these two indicators. The nomogram had a moderate ability to predict prognosis of patients with HCC and could also accurately predict the prognosis of these patients in the external validation dataset. Using the nomogram model we constructed, we predicted the 1-year, 2-year, and 3-year survival rates of each patient with HCC based on the expression of these four lncRNAs in the tumor tissue of each patient and the tumor stage. The higher the overall score, the worse the 1-year, 2-year, and 3-year survival rates of patients with HCC. This is based only on the results of the public database, and multi-center data are needed for verification in the future. Suppose the reliability of the model is verified in a multi-center cohort, the corresponding lncRNA panel could be developed for clinical applications to provide medical advice. It can be used to predict the prognosis of patients with liver cancer.ZEB1-AS1 is transcribed by the bidirectional promoter of ZEB1. In our study, ZEB1-AS1 was significantly increased in liver cancer tissues and significantly associated with poor prognosis in patients with liver cancer. in vitro studies further confirmed that silencing MIR210HG significantly reduced the proliferation, invasion, and migration capabilities of liver cancer cell lines. These results are consistent with those of our research.MIR210HG is transcribed from ENSG00000247095.2 present on human chromosome 11p15.5. MIR210HG is elevated in a variety of tumors such as cervical cancer, non-small cell lung cancer, breast cancer, colorectal adenocarcinoma, pancreatic cancer, liver cancer, osteosarcoma, and glioma . For exaSince BACE1-AS was found to be abnormally expressed in Alzheimer’s disease by SNHG3 plays a vital role in the occurrence and development of many tumors, including liver cancer . In our Although our study obtained a novel m6A-related lncRNA signature for predicting prognosis of patients with HCC, it also has some limitations. First, the interactions between m6A regulatory factors and lncRNAs should be experimentally verified. Second, because of the limited number of HCC datasets with prognostic information and lncRNA gene symbols, we used only the ICGC dataset for external verification.In conclusion, we constructed an m6A-related lncRNA signature to predict the prognosis of HCC patients and also constructed a predictive nomogram that can more accurately predict the prognosis of HCC patients and provide guidance for individualized treatment of HCC.


Encapsulation is a versatile technique used to protect sensitive bioactive compounds under gastrointestinal conditions. In this study, nanoencapsulation of chlorogenic acid into the apple seed protein matrix was performed using the green technique ultrasonication to protect it from harsh gastric conditions and increase its biological activity and bioavailability upon digestion. Both nano (Nano-Chl) and native capsules (NT-Chl) were characterised by particle size, charge, structure, and morphology. The encapsulation efficiency, release behaviour, antioxidant and antidiabetic properties were also evaluated. The experimental results show that the particle size of the NT-Chl and Nano-Chl was found in the range of 1.4 µm to 708 nm. The encapsulation efficiency was found to be 69% and 80% for NT-Chl and Nano-Chl, respectively. Furthermore, an in vitro digestion study revealed that Nano-Chl showed controlled-release behaviour under simulated intestinal conditions in comparison to NT-Chl. Moreover, Nano-Chl showed enhanced antioxidant and antidiabetic activity in comparison to NT-Chl after simulated digestion. It was concluded that the protein from apple seeds could be utilised as a functional ingredient itself or as a wall material for the encapsulation of sensitive bioactive compounds. Furthermore, these encapsulated particles can be fortified into different food formulations for the development of functional food. Chlorogenic acid is a polyphenolic compound existing widely in spices, fruits, vegetables, green coffee, etc., with green coffee being the richest source. . It exhiThe present study was planned to nanoencapsulate chlorogenic acid into the apple protein as a wall material and analyse its nutraceutical potential, release behaviour, and encapsulation efficiency. Furthermore, this study opens ways to utilise apple seed protein itself as a functional ingredient or as a wall material for encapsulating bioactive ingredients. Moreover, developed native- and nano-apple protein-loaded chlorogenic acids can have incredible food and pharmaceutical uses as protein supplements.Apple seeds were procured from FIL Industries, Pvt. Ltd., Srinagar, Jammu and Kashmir, India. The samples were cleaned, dried, dehulled, and then stored at 4 °C in airtight containers. The apple flour obtained was coarsely ground using a domestic blender. The chemicals and enzymes, such as α-amylase, α-glucosidase, pancreatin, pepsin, bile salts, DPPH, etc., used in this study were purchased from Sigma-Aldrich .g for 10 min. The supernatant obtained was precipitated at the isoelectric point (pH 4.5) using 1 N HCl and re-centrifuged to recover the protein (pellet). The protein was freeze-dried and stored at −4 °C till use. The protein content in the freeze-dried sample was estimated to be 95.4 ± 0.54% on a dry-weight-basis (AOAC 1997) [Briefly, defatted apple seed flour was dissolved in deionised water (pH 9–10.5) and stirred for 2 h at room temperature (25 °C) for the complete solubilisation of proteins . This waAC 1997) .Apple seed protein nanoparticles were prepared following the method of Shah et al. . BrieflyThe encapsulation of chlorogenic acid into protein nanoparticles was performed following the procedure of Shah et al. . Both nag for 10 min. The supernatant was discarded, and the swollen mass (pellet) was removed, slightly dried, and reweighed. The swelling index (SI) was calculated as follows:s = the weight of the swollen sample and Md = the weight of the dried sample.The swelling index (SI) of the encapsulated samples was conducted following the method of Shah et al. . EncapsuThe particle characteristics such as particle size (PI), polydispersity index (PDI), and zeta potential (ZP) were evaluated using dynamic light scattering (DLS) . Both native- (NT-Chl) and nanoencapsulated (Nano-Chl) capsules were dispersed in distilled water (0.02%), followed by sonication for 10 min at 20 kHz in a sonicator bath. The sonicated solution was then analysed for particle size (PI), polydispersity index (PDI), and zeta potential (ZP).The microstructural analysis of the samples (NT-Chl and Nano-Chl) was visualised using a scanning electron microscope  at a voltage of 15 kV. The sample was placed on the aluminium stubs coated with gold and investigated for morphological characteristics under vacuum conditions.−1 at 2 cm−1 of resolution.The structural changes in the NT-Chl and Nano-Chl capsules were evaluated using FTIR . The FTIR spectra were collected at wavelengths ranging from 4000–400 cmg for 10 min, and the absorbance of the supernatant was taken at 327 nm. EE was calculated by using Equation (1) y = 0.029x + 0.014 R2 = 0.904:The encapsulation efficiency (EE) of the capsules was performed following the method of Gani et al. . Brieflyg for 5 min, and the release of chlorogenic acid was observed at 327 nm. The pellet was resuspended in 10 mL of simulated gastric juice containing pepsin (3 g/L) prepared in sterile NaCl solution (6g/L) and after constant stirring, aliquot (1 mL) was collected after 30 and 60 min of incubation. The mixture was centrifuged at 1500× g for 5 min. The supernatant was collected and release of cholorognic acid was observed under simulated gastric conditions. The recovered pellet from gastric phase was subjected to simulated intestinal conditions using pancreatin (10 g/L) and bile salts (3 g/L) prepared in phosphate buffer solution (pH 7.5) and incubated at 37 °C with gentle stirring. The whole reaction mixture was centrifuged at 5000× g for 5 min at the interval of 30, 60, and 120 min to evaluate the release of chlorogenic acid from the protein matrix under intestinal conditions. The amount of chlorogenic acid (%) released was measured and calculated as described in The in vitro release study of encapsulated capsules was studied under simulated gastrointestinal conditions following the method of Jhan et al. . NT-Chl g for 10 min and supernatant was stored at 4 °C for antioxidant and antidiabetic analysis.The encapsulated capsules (NT-Chl and Nano-Chl) were evaluated for bioactivity, such as antidiabetic and antioxidant properties, to study the effect of the encapsulation process on the retention of chlorogenic acid into apple seed protein matrix after in vitro simulated gastrointestinal digestion. Briefly, 100 mg of NT-Chl and Nano-Chl were dissolved in simulated gastric conditions (pepsin) and incubated at 37 °C for 1 h. This was followed by exposure to simulated intestinal conditions  and re-incubated at 37 °C for 2 h. The mixture was centrifuged at 4000× DPPH ActivityFree radical scavenging activity of the samples was performed following the method of Ashraf et al. . BrieflyPercent inhibition was calculated using Equation (1):Metal Chelating2 (2 mM) was incubated for 30 min at room temperature. After incubation 0.25 mL ferrozine (5 mM) was added and incubated for 30 min. The absorbance of the sample mixture was taken at 700 nm, and % inhibition was calculated using Equation (1).The metal-chelating activity of the encapsulated capsules was evaluated following the procedure of Ashraf et al. . The reaα-Amylase Inhibitory Activityo signifies the absorbance of the negative control, and Ap signifies the absorbance of the positive control.The α-amylase inhibitory activity was performed following the method of Ashraf et al. . The reaα-Glucosidase Inhibitory ActivityBriefly, the incubation of the reaction mixture consisting of sample (25 μL), 0.1 M sodium phosphate  and 25 μL α-glucosidase solution for 5 min was performed at 37 °C. Then, 25 μL of PNPG (15 mM) was added to start the enzymatic reaction and again incubated for 40 min. An amount of 100 μL sodium carbonate (0.2 M) was added to terminate the reaction. The hydrolysis of substrate by α-glucosidase was calculated at 405 nm. Inhibition (%) was evaluated using Equation (2).t-test at 95% significance level was used to assess the difference between the mean using commercial statistical software (IBMSPSS statistics 21). All the experiments were carried out in triplicates. The data were presented as mean ± standard deviation. Students p < 0.05) increased upon the encapsulation process as presented in The encapsulated capsules were characterised by size, polydispersity index, and surface charge using dynamic light scattering. Particle size is an important characteristic for understanding the behaviour of the encapsulated capsules, such as their encapsulation efficiency, release kinetics, solubility, and functionality . The parZeta potential (ZP) represents the surface charge of the particles and provides information about attractive and repulsive forces between the particles. It influences the kinetic release and biological fate of nanoparticles . As can Swelling behaviour is an important parameter that defines the ability of an encapsulant to entrap the sensitive ingredient under the simulated gastrointestinal tract conditions. The encapsulated capsules were rehydrated in a phosphate buffer solution at pH values 3 and 7.5 to mimic the gastrointestinal environment . It can p < 0.05). The enhanced EE of the Nano-Chl could be ascribed to the nanosized particles used for encapsulation. Fine particle sizes have good EE because of their ability to produce a film around the bioactive ingredient; hence, there is higher retention of chlorogenic acid [Encapsulation efficiency (EE) is an essential feature that is defined as the fraction of bioactive compound entrapped inside the biopolymer matrix. The EE of NT-Chl and Nano-Chl was found to be 69.1% and 80.3%, respectively, as shown in nic acid ,19. Ariynic acid  reportedThe morphological characteristics of NT-Chl and Nano-Chl were evaluated using SEM. NT-Chl showed a crystalline flake-like structure with greater dimensions, whereas Nano-Chl showed smaller, regular, and homogeneous structures. The ultrasonication process resulted in the disintegration of larger flake-like structures into finer regular structures  in the n−1 representing OH vibrations. The peak at 1500–1750 cm−1 can be related to the C=C vibrations of an aromatic ring and 1250 cm−1 to the C-O broadening. The infrared spectra of apple seed protein (N-Protein and US-Protein) represent a wide band stretching of peaks from 3000–3400 cm−1 corresponding to the existence of amide A (N-H bonds), 1600–1750 cm−1 corresponding to amide I, 1400–1570 cm−1 corresponding to amide II, and 1050–1200 cm−1 corresponding to amide III regions, which is due to the C=O stretching and combination of C-N and N-H bending vibrations [The FTIR analysis of N-Protein, US-Protein, NT-Chl, Nano-Chl, and chlorogenic acid is represented in brations . However3, rutin, and catechin from nanoencapsulated capsules. Moreover, Mohammadian et al. [The purpose of the nanoencapsulation was to limit the degradation and abrupt release of chlorogenic acid in the simulated gastrointestinal conditions and retain its bioactivity at the targeted site . Chlorogn et al.  describeThe antidiabetic activity of the nanoencapsulated capsules was evaluated using in vitro α-amylase and α-glucosidase inhibition activity. This involves the hydrolysis of starch molecules into sugar. Hence, inhibitors to this enzyme inhibit the digestion and absorption of dietary starches by the body, whereas α-glucosidase involves the hydrolysis of disaccharides to monosaccharides and the inhibition of α-glucosidase controls blood glucose and, thus, decreases postprandial plasma glucose levels in diabetics. The percent inhibition value of α- amylase and α-glucosidase varied significantly before and after digestion, as shown in p < 0.05) before and after digestion. Before digestion, it was found to be 64.7–78.1%; however, after digestion, it was found to be 60.3–67.23%. The hydroxyl groups and higher electron density on chlorogenic acid will easily provide electron for stabilizing free radical. However, in the case of metal-chelating activity, chelating agents inactivate metal ions and potentially inhibit the metal-dependent processes providing antioxidant activity. The percent inhibition activity of NT-Chl and Nano-Chl was evaluated before and after digestion. Before digestion, it was found to be in the range of 69.1–81.6%, and after digestion, it was found to be in the range of 65.2–69.3%. Overall, the results reveal that the nanoencapsulation process prevented the deterioration of chlorogenic acid, and a good amount of chlorogenic acid was retained at the targeted site. Nallamuthu et al. [The antioxidant activity of NT-Chl and Nano-Chl was evaluated against free radical scavenging activity (DPPH) and metal-chelating activity before and after in vitro digestion, as shown in This study involves the encapsulation process of chlorogenic acid, which showed enhanced bioavailability and bioactivity and provided a comparison between the micro- and nanoencapsulated systems of apple seed protein loaded with chlorogenic acid. Nano-Chl provided good EE in comparison to NT-Chl with sustainable releasing properties. The Nano-Chl also showed higher antioxidant and antidiabetic properties as compared to NT-Chl indicating that nano-delivery systems can be incorporated into different food and pharmaceutical uses for the increased delivery of chlorogenic acid. Moreover, this study provides an avenue for the utilisation of agro-waste as a novel source of protein for the encapsulation of sensitive bioactive compounds.


Streptococcus mutans and Candida albicans), typical of dental caries found in children. The prepared pSi microparticles were roughly cubic in structure with 70–75% porosity, to which the TC (pSi-TC) was loaded with about 45% efficiency. The pSi-TC particles exhibited a controlled release of the cargo over a 14-day period. Notably, pSi-TC significantly inhibited biofilms, specifically downregulating the glucan synthesis pathways, leading to reduced adhesion to the substrate. Acid production, a vital virulent trait for caries development, was also hindered by pSi-TC. This pioneering study highlights the potential to develop the novel pSi-TC as a dental caries-preventive material. Dental caries, a preventable disease, is caused by highly-adherent, acid-producing biofilms composed of bacteria and yeasts. Current caries-preventive approaches are ineffective in controlling biofilm development. Recent studies demonstrate definite advantages in using natural compounds such as trans-cinnamaldehyde in thwarting biofilm assembly, and yet, the remarkable difficulty in delivering such hydrophobic bioactive molecules prevents further development. To address this critical challenge, we have developed an innovative platform composed of components with a proven track record of safety. We fabricated and thoroughly characterised porous silicon (pSi) microparticles to carry and deliver the natural phenyl propanoid trans-cinnamaldehyde (TC). We investigated its effects on preventing the development of cross-kingdom biofilms ( The oral cavity is home to several bacteria and fungi, which include both beneficial and harmful species. However, the formation of a highly adherent plaque biofilm with an increase in pathogenic species (dysbiosis) results in costly diseases such as dental caries ,2. FluorStreptococcus mutans [Enterococcus faecalis [Streptococcus mutans and Enterococcus faecalis [Trans-Cinnamaldehyde (TC) is a bioactive flavonoid with antimicrobial activity against several bacterial and fungal species ,11,12,13faecalis ,11,12,13Micro and nanoparticles are efficient methods of increasing the availability of water-insoluble or sparingly soluble bioactive molecules. Such particles can be fine-tuned to allow for the controlled release of the drug and elicit target-specific action, while dramatically reducing the concentration of the drugs required for their action . Porous Streptococcus mutans is the primary driver of the caries process as it produces acids which cause demineralization of the dental hard tissues [S. mutans and the yeast Candida albicans results in an increase in the cariogenic virulence of plaque [ tissues . Being a tissues . Such a f plaque ,22,23 anf plaque . While sS. mutans + C. albicans). We tested the hypothesis that TC-eluting pSi particles (pSi-TC) can prevent the development of biofilms and inhibit its caries-related virulence phenotype (acid production).Therefore, the aim of this study was to encapsulate TC in pSi particles and examine its efficiency in preventing the development of cross-kingdom biofilms  to create a porous structure. This porous layer was detached from the bulk wafer with a second etching in 3.1% HF in ethanol, at a current intensity of 4 mA/cm2 for 4 min. The detached layer was rinsed with ethanol and subjected to sonication for 5 min at 25 Hz in an ultrasonic bath. All particles were thermally oxidized for 1 h at 400 °C to slow the particle resorption in aqueous solution.Porous silicon wafers were prepared by the electrochemical etching of crystalline silicon wafers  in a custom-made Teflon cell. The etching was performed under a current density of 200 mA/cmThe microparticles were sterilized with 70% ethanol for 10 min before drying under sterile airflow. The pSi particles were analyzed by scanning electron microscopy (SEM)  to determine the particle size and pore diameter. An acceleration voltage of 20.00 kV was used at a pressure of 0.5 Torr. Global porosity was determined by interferometric reflectance spectroscopy (IRS), using white light at normal incidence, with reflected light collected by a charge-coupled device (CCD) spectrometer. Particle oxidation was controlled by Raman spectroscopy using a Witec Confocal Raman Microscope System alpha 300R  to register the Raman spectra. Excitation in the confocal Raman microscopy was assured by a frequency-doubled Nd: YAG laser  at 532 nm wavelength .−1 with 30 scans per sample. The data acquisition was performed by OPUS 7.5  software.We performed Fourier Transformed Infrared spectroscopy (FTIR) to determine if there was any chemical interaction between the pSi and TC. The TC-loaded pSi particles were taken and washed with deionized water three times and then dried the obtained particles for analysis. The spectra were collected over a range of 4000–400 cmv/v) in ethanol was used. After 24 h of incubation in loading solution, the pSi particles were rinsed in ethanol and stored in 70% ethanol solution to prevent particle degradation. Loading efficiency was assessed by UV-visible spectroscopy at a wavelength of 290 nm.The pSi particles were loaded with trans-cinnamaldehyde  by passive soaking for 24 h under gentle shaking at room temperature. For loading, a 1:1 dilution of trans-cinnamaldehyde (2adjusted (R2adj) values. The model that exhibited the R2adj value closest to one was considered the best-fitted kinetic model [To follow the trans-cinnamaldehyde release, the particles were dried to remove the ethanol and then incubated in Phosphate Buffer Saline (PBS)  at 37 °C. Release experiments were conducted by incubating 25 mg of TC loaded with 10 mL of PBS, i.e., 2.5 mg particles/mL of PBS. Supernatants were collected after one, four, eight, and fourteen days. The recovered samples were analyzed by High-Performance Liquid Chromatography  with the following parameters: a 254 nm UV detector and an elution with 0.1 formic acid solution in acetonitrile at 0.5 mL/min debit. Various concentrations of TC in ethanol were used for calibration to determine the retention time (RT). For the elucidation of various kinetics models, they were tested for best fit after applying data obtained from the release study. The best-fit kinetic model was selected by comparing their RStreptococcus mutans UA159 and Candida albicans ATCC 90028 were acquired from the American Type Culture Collection (ATCC). S. mutans was maintained in Columbia Blood Agar (CBA) at 37 °C in anaerobic conditions , while C. albicans was maintained in Sabouraud Dextrose Agar (SDA) at 37 °C in aerobic conditions. For all the experiments, S. mutans and C. albicans were grown overnight in Tryptic Soy Broth (TSB) at 37 °C in a 5% CO2 incubator. Before each experiment, the microbial count was adjusted to 2 × 106 for S. mutans and 2 × 104 for C. albicans [albicans .S. mutans and C. albicans monocultures was determined by the broth microdilution assay [2 incubator for 24 h. The microbial growth was determined by measuring the absorbance (OD595) of the planktonic cells using a DTX 880 Multimode Detector .The effect of pSi-TC on the growth of on assay . The stoS. mutans and C. albicans and incubated at 37 °C in a 5% CO2 incubator for 24 h. Then, the planktonic cells were removed, and the biofilms were carefully washed twice with PBS and stained with 0.1% crystal violet. After washing with PBS, the CV stain absorbed by the biofilm biomass was retained by adding 95% ethanol, transferred to another 96-well plate, and the absorbance (OD570) was measured using a multimode detector.The effect of pSi-TC on biofilm formation was determined directly by the well-established Crystal Violet (CV) assay. pSi-TC (100 µL) was added to TSB + 1% sucrose with 10 µL of the standard cell suspension for each of S. mutans and C. albicans in the pSi-TC treated biofilms. Dual-species biofilms were developed on sterile hydroxyapatite discs  in the presence of 0.25 mg/mL of pSi-TC based on the results of the aforementioned experiment. The treated and untreated biofilms were mechanically disrupted and dispersed in 1 mL of PBS by uniform vortexing for 1 min.The plate count method was used to determine the proportion of C. albicans and S. mutans, respectively, and incubated in an anaerobic chamber and aerobic incubator, respectively, at 37 °C for 24 h. The number of viable cells was determined by counting the Colony Forming Units (CFU), which were transformed into log10 values.The resultant microbial suspension was then serially diluted in TSB, and a 50 µL aliquot was plated on Sabouraud Dextrose Agar (SDA) and Columbia Blood Agar (CBA) for v/v).Dual-species biofilms were developed in the presence of 0.25 mg/mL of pSi-TC. Then, the pH of the spent media was determined using a pH electrode , as described previously . Prior tTo directly visualize the biofilm-preventive effect of pSi-TC, biofilms were developed in the presence of pSi-TC. After 24 h of incubation, the discs were washed with PBS and fixed with 2.5% glutaraldehyde, following dehydration in ethanol series . Then, the discs were gold sputter-coated and observed using SEM (Hitachi VP-SEM SU1510) to assess the biofilm architecture .g for 10 min.The effect of pSi-TC on the biofilm and virulence-related genes was studied using Quantitative real-time Polymerase Chain Reaction analysis (qRT-PCR). Biofilms were developed in the presence of pSi-TC for 24 h. Untreated biofilms served as the negative control. After incubation, the planktonic and loosely adhered cells were removed by washing the biofilms twice with PBS. The biofilms were then removed by scraping and centrifuged at 10,000× TM cDNA synthesis kit was used to reverse transcribe RNA to cDNA, following which the qRT-PCR analysis was performed. The primers used for C. albicans and S. mutans are listed in S. mutans and C. albicans respectively. The 2-ΔΔCT method was used for calculating the gene expression changes relative to the control [RNA was extracted as per the manufacturer’s instructions and the quantity of RNA was assessed using Nanodrop. An iScript control .p < 0.05 was considered statistically significant.Statistical analysis of the data was performed using Prism version 8.0.2 . One-way ANOVA multiple comparison analysis was performed to compare the significance between the control and treatment groups, while the Mann-Whitney U-test was applied to the compare the mean pH values between the groups. The antimicrobial and anti-biofilm efficacy of essential oils is masked in low concentrations as they are relatively unstable and easily degradable . Thus, t−1 (scissoring vibration) and at 468 cm−1 (bending vibration)  . This oxw/w). In addition, the maximal possible loading determined according to porosity (70–75%), molecular weight (MWCinnam = 132 g/mol), density (dCinnam = 1.05), and dilution of pSi-TC particles. According to this, the maximal theoretical loading was found to be 0.3 g TC/g of particles.The assessment of TC loading by UV-Vis spectroscopy revealed an average TC loading of 45% in pSi particles . The concentration of cinnamic acid increased over time, while the cinnamaldehyde concentration decreased. Thus, the drug release kinetics showed a sustained and controlled release of TC from the pSi over a period of 14 days. Nevertheless, from the kinetics modelling (2adj) = 0.998, n = 0.228, which confirmed that the pSi-TC formulation followed a pseudo-Fickian transport [The TC concentrations recovered at the different time points were Codelling  it has bransport .−1 is attributed to Si-O-Si asymmetric stretching; the sharp peaks at 796 cm−1, 964.99 cm−1, and 470.82 cm−1 correspond to the O-Si bond, observed in the spectrum of pristine pSi particles [−1 is ascribed to the aromatic C–H bond; 2924 cm−1 due to the =C–H bond; 2854 cm−1 is assigned to the C–H bond of the carbonyl groups [−1 and 2924 cm−1 peaks were suppressed because of the high-intensity pSi particles  in comparison to the control, whereas in S. mutans and C. albicans mono-species biofilms, a 50% biomass reduction was observed at concentrations of 0.25 mg/mL and 0.009 mg/mL, respectively  in the pH of the spent media, indicating decreased acid production within biofilms that were exposed to pSi-TC .The concentration of TC in 0.25 mg/mL of pSi-TC is 87.5 µg/mL. This is an important finding since a six-fold reduction in the TC concentration was sufficient to inhibit dual- and mono-species biofilms of eviously . The impeviously ,16. Therp < 0.05) the S. mutans genes that regulate inter-species communication and biofilm development (~1.5 fold) (LuxS), genes governing heat and acid-induced stress (>2.5 fold) (DnaK and AtpD) [Nox1) [S. mutans and controls interspecies biofilm formation, acid tolerance, and stress tolerance mechanisms [p < 0.05), corroborating strongly with our phenotypic results.To understand the underlying mechanism of these biofilm-inhibitory and acid production-reducing responses of pSi-TC, gene expression analysis was performed . qRT-PCRnd AtpD) , and oxi) [Nox1) . The Luxchanisms , which wGtfB and GtfC contribute markedly to glucan production, which forms the bulk of the EPS matrix. In addition, these genes also regulate biofilm development and facilitate bacterial adhesion to the salivary pellicle and microcolony formation, which further enhance the adhesion of C. albicans to the biofilms [GtfB mutant strain of S. mutans was unable to synergize with C. albicans, preventing the development of serious carious lesions in animal models [C. albicans also produces glucans, which are regulated by Bgl2, Phr1 and Phr2 genes, all of which were significantly downregulated by pSi-TC [C. albicans also activates S. mutans GtfB to produce more glucans, forming a well-organized biofilm model [C. albicans cells by >4 log in dual-species biofilms, it is likely that pSi-TC prevented the synergism between S. mutans and C. albicans by inhibiting the glucan synthesis which leads to decreased adhesion to the substrate.Notably, pSi-TC also significantly downregulated the genes that encode for Glucosyl transferases (>2.5 fold) . The genbiofilms ,47. In fl models . Furthery pSi-TC ,49,50. Clm model . By reduWhile we did not characterise the cytotoxicity in this study, there is ample evidence from us and others, to support that pSi and TC are both remarkably safe compounds. The biocompatibility of pSi to several cell types has been demonstrated previously by us ,52,53 anA limitation of this work was that we used a representative cross-kingdom biofilm that is typical of early childhood caries. While the use of microbes from two kingdoms strengthens the validity of the results, it does not recapitulate the complexity of the oral ecosystem and the caries microbiome, considering that oral biofilm houses more than 700 microbial species. Thus, further studies will interrogate the effects of pSi-TC on complex biofilm models in the presence of salivary conditions to better replicate the host environment. Such studies will provide insights into how pSi-TC can modulate microbial ecology.Porous silicon particles are an innovative and effective approach to carrying and releasing TC.S. mutans and C. albicans and its acid production.pSi-TC successfully inhibited the development of cross-kingdom biofilms of This pioneering in vitro study demonstrated that:The newly developed pSi-TC holds remarkable promise to be further developed as a caries-preventive agent.


This study aimed to investigate the immediate and continual perturbation to the gut microbiota of offspring in the weeks post-weaning and how these may be modulated by treating pregnant C57BL/6J dams with antibiotics (ABX). We used a broad-spectrum antibiotic cocktail consisting of ampicillin 1 mg/mL, neomycin 1 mg/mL, and vancomycin 0.5 mg/mL, or vancomycin 0.5 mg/mL alone, administered ad-lib orally to dams via drinking water during gestation and stopped after delivery. We analyzed the gut microbiota of offspring, cytokine profiles in circulation, and the brain to determine if there was evidence of a gut-immune-brain connection. Computationally predicted metabolic pathways were calculated from 16s rRNA sequencing data. ABX treatment can negatively affect the gut microbiota, including reduced diversity, altered metabolic activity, and immune function. We show that the maternal ABX-treatment continues to alter the offspring’s gut microbiota diversity, composition, and metabolic pathways after weaning, with the most significant differences evident in 5-week-olds as opposed to 4-week-olds. Lower levels of chemokines and inflammatory cytokines, such as interleukin (IL)-1α and IL-2, are also seen in the periphery and brains of offspring, respectively. In conclusion, this study shows maternal antibiotic administration alters gut microbiome profiles in offspring, which undergoes a continuous transformation, from week to week, at an early age after weaning.  Streptococcus (GBS), yeast infection, bacterial vaginosis and other sexually transmitted diseases, and urinary tract infections [When major viral pandemics occur, such as SARS, swine flu, MERS, Ebola, Zika, and now COVID-19, there is an increase in prescribing antibiotics (ABX) for use as therapeutic and prophylactic treatments for bacterial coinfection or secondary infection ,3,4. Thefections ,19,20. Pfections ,22. TherTreatment with broad-spectrum antibiotics, such as penicillin plus gentamicin, is considered standard for maternal infections . These aThe gut microbiota directly influences the maturation of the immune system, where deviations from the normal development of the gut microbiota alter outcomes of immune development and potentially increase susceptibility in offspring to various diseases later in life ,31. As tA considerable number of studies regarding maternal antibiotic treatment during prenatal and early postnatal periods have shown changes in the gut microbiota and altered immune profiles, increasing the risk of disease and morbidity over time . Our stup ≤ 0.0001) compared to the naïve group (p ≤ 0.0001) The size of the cecum in the vancomycin group did not differ from controls. The gut microbiota was analyzed by Illumina 16S rRNA based sequencing of bacterial DNA extracted from fecal pellets. Bacterial taxonomy was used for classification, where the abundance of the amplicon sequence variable (ASVs), also referred to as “observed features”, were determined. The maternal ABX-cocktail treatment led to a decrease in observed features  (p = 0.0011 and p = 0.0909), at 4 and 5 weeks, respectively. Following LPS challenge, differences in the observed features in all groups were seen, with the most significant differences occurring in the vancomycin groups (p ≤ 0.0001). When the vancomycin group was challenged with LPS, observed features were decreased significantly at 5 weeks  compared to the naïve offspring challenged with LPS. When challenged with LPS, there was an increase in observed features in the ABX-cocktail . When comparing the 4-week-olds to the 5-week-olds, after LPS challenge in the ABX-cocktail treatment group, observed features were significantly different . In this study, we investigated the impact of maternal ABX-treatment on the gut microbiota of prepubescent mice offspring, at weeks 4 and 5, corresponding to the prepubescent stage. Lipopolysaccharide, a cell wall constituent of Gram-negative bacteria that is abundant in the GI tract, was injected intraperitoneally to induce a systemic immune response in prepubescent mice, to assess the influence of prenatal ABX treatment on immune responses. The maternal ABX-treatment had no significant effects on body weight group  when comve group . After Lctively) A. In conp = 0.9948 and p = 0.0019, at 4 and 5 weeks, respectively) . Finally, when comparing the 4-week-olds to the 5-week-olds, after the LPS challenge, there was a significant increase in diversity in the ABX-cocktail group . Next, to determine the impact on β diversity, cluster analysis with Bray–Curtis was performed to assess dissimilarity between groups. Maternal ABX-treatment led to a clear shift in β-diversity as seen by the analysis of community structures. The ABX-cocktail group clusters further away from the naïve controls at baseline but are clustered more closely after the LPS challenge  B. The vahallenge C. In genFirmicutes and Bacteroidetes were the dominant phyla accounting for most of the assigned sequences, while Proteobacteria and Actinobacteria had relatively low abundances  in the 4-week-olds as compared to the naïve controls . These changes were less pronounced at 5 weeks. In the ABX treatment group, there was a decrease in Firmicutes and increased Bacteroidetes at 5 weeks compared to the controls . It has previously been shown that an ABX-induced decrease in diversity resulted in an increase in the Firmicutes to Bacteroidetes (F/B) ratio [To further characterize the dysbiotic phenotype of the prepubescent offspring of ABX-treated dams, we examined how the altered α/β diversity contributes to the taxonomic community structure. Only four bacteria phyla were found in all groups; undances . The matcontrols A,B. ChanB) ratio . IncreasFirmicutes and Bacteroidetes only after LPS . Additionally, after the LPS challenge, a significant difference was seen in the 5-week-olds from the vancomycin group, which was reduced compared to vancomycin without LPS stimulation , and the vancomycin group challenged with LPS was lower than the naïve controls challenged with LPS (mean relative abundance Bacteroidetes 25% versus <1% p = 0.0405 at 5 weeks). When comparing the 4-week-olds to the 5-week-olds, the ABX-cocktail led to a significant difference in Actinobacteria and Proteobacteria populations were less abundant, resulting in less variation between groups and no significant differences at either age when comparing the naive controls to the ABX-cocktail and vancomycin groups at baseline. However, after the LPS challenge, the 4-week-olds did show significant differences from their baseline  and after LPS challenge in the group  . In the ABX-cocktail group there was a difference after the LPS challenge at the two time points in the Proteobacteria .4 weeks) C,D. At 5Lactobacillus spp. and Muribaculaceae family), while other genera appeared to colonize more or less abundantly (Bacteroides and Parasutterella spp.), or only occurred after the LPS challenge (Enterococcus and Enterobacteriaceae spp.)  , but was absent at 5 weeks   . Regardl4 weeks) A. No dif 0.0001) B.Muribaculaceae family is the most abundant of the phyla Bacteroidetes. The populations of Muribaculaceae family in both naïve controls and ABX-cocktail group were decreased following the LPS challenge in the 4-week-old  . Muribaculaceae family in the vancomycin following LPS group decreased at 5 weeks . Bacteroides spp. also from the phyla Bacteroidetes, were depleted entirely at baseline in the ABX-cocktail group at 4 and 5 weeks; however, after LPS challenge in the ABX group at 5 weeks Bacteroides spp. was significantly increased compared to ABX without LPS challenge (p ≤ 0.0001) ( 0.0044) C. At 5 w 0.0001) D. Parasutterella spp. belongs to the phyla Proteobacteria and was only seen in the vancomycin group at baseline at 4 weeks (p = 0.0139) and also populated in controls following the LPS challenge while the vancomycin group had the opposite effect (p = 0.0078), compared with naïve controls  and compared with the naïve group following the LPS challenge (p ≤ 0.0002), at 4 weeks  effect size (LEfSe) analysis of MetaCyc pathways was used to determine where several metabolic pathways were biologically significantly altered, with the majority coming from the vancomycin groups in the 5-week-olds .The ABX-cocktail group had significant differences in pathways associated with glycolysis V and allantoin degradation IV. In contrast, the microbiota in the vancomycin group had differences in pyridoxal 5’-phosphate and heme biosynthesis in the 4-week-olds A. The guIn summary, The LDA effective size (LEfSe) was performed to identify MetaCyc pathways with statistically differential abundance between the ABX-cocktail and vancomycin group, where the 5-week-olds from the vancomycin group had carbohydrate biosynthesis, amino acid biosynthesis, cofactor, carrier, and vitamin biosynthesis, secondary metabolites biosynthesis, and other biosynthesis pathways with LDA score greater than 2.0. In contrast, the ABX-cocktail only had nucleotide and nucleoside biosynthesis and fatty acid and lipid biosynthesis pathways with LDA score greater than 2.0. The effect of maternal ABX-treatment on metabolism-related pathways were more abundant in the vancomycin group than in the ABX-cocktail group. However, based on these predicted pathways, there are alterations in 4-weeks-olds, though to a lesser extent, we see that antibiotics significantly alter essential metabolic pathways in the prepubescent stage. Combined with altered diversity and taxonomic population shifts, these findings paint a picture of gut microbiota dysbiosis . Therefop = 0.0012 and p = 0.0431), while the vancomycin treatment had no significant effect on either IL-1α and MIP-2, also known as CXCL2 . This effect was not seen in the maternal ABX-treatment offspring following the LPS challenge. There were no significant differences seen in the 4-week-olds  at baseline without LPS challenge, and IL-12(p40) following LPS challenge (p = 0.0353) in the 5-week-olds (p = 0.0030), and IL-17 (p = 0.0048) than at 4-week-olds . The mice were given a week to acclimate to the vivarium before breeding started. The dams were mated between 6–8 weeks of age, with one male and two females per cage. The embryonic (E) day was determined by the presence of a vaginal plug marking E0.5, and the males were then removed from the cage. Dams were either untreated controls (naïve group) or treated orally via drinking water from E14.5 until birth, i.e., acutely (5 days) with either vancomycin (0.5 mg/mL)  alone (vancomycin group), or ampicillin (1 mg/mL), neomycin (1 mg/mL)  and vancomycin (0.5 mg/mL) in combination (ABX-cocktail group). This regimen is a modification to is consistent with standard antibiotic administration used in the protocol described by Rakoff-Nahoum et al. . Here, aEscherichia coli O55:B5  into 4-week-old and 5-week-old prepubescent mice. An equivalent amount of phosphate-buffered saline (PBS) was given to the controls. The fecal samples were collected 24 h post-injections, and animals were euthanized for serum and brain tissue collections. The peak brain immune response after the LPS challenge is seen at this time. Additionally, it also provided time for gut microbiota changes to the immune stimulation. Fecal samples were collected from individually isolated mice that were placed in sterile cages to collect fecal pellets. The collected samples were stored directly on dry ice before being transferred to −80 °C until analysis. Animals were then anesthetized with isoflurane. Blood was drawn into an uncoated capillary tube via a nick in the cardiac artery. The blood was transferred to a tube and allowed to clot at room temperature before placing it on ice. After a 10 min centrifugation at 4 °C and 13,000× g, blood serum was collected and stored at −80 °C until analysis. Following blood collection, brains were perfused with HBSS through the left ventricle; then, the extracted brain was hemi-sectioned for protein extraction. The cecum was also removed from the gut for weight measurements. All tissue samples were immediately frozen on dry ice and then stored at −80 °C until analysis.A systemic immune response was initiated by an intraperitoneal (IP) injection with 5 mg/kg of body weight lipopolysaccharide (LPS) from For the microbiota analysis, bacterial DNA was extracted from fecal samples of the naïve, ABX-cocktail and vancomycin groups at 4 and 5 weeks. Using barcoded primers, the 16S rRNA amplicon sequencing of the V4 region was performed using the MiSeq platform . In brief, the Illumina demultiplexing, merging of the reads and trimming of the barcodes and primers, and the QC were performed by the UC Davis sequencing core. FastQ files were then analyzed using the QIIME2 software package v. 2021.2 . DADA2 pg for 10 min at 4 °C) to precipitate insoluble materials. The hippocampus was processed similarly to the whole brain.For the cytokine analysis, serum was collected from the clotted blood samples. Brain samples were collected from mice and immediately stored at −80 °C until use. Individual brain samples were suspended in PBS with a protease inhibitor cocktail , then physically disrupted by vortexing thoroughly, followed by brief sonication. The disrupted pellets were centrifuged . The quantification of cytokines was determined using mouse reactive Milliplex™ multiplexing bead immunoassays . Which allows for the simulatinuse detection of: G-CSF, GM-CSF, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, MCP-1, MIP-1α, MIP-1β MIP-2, RANTES, and TNF-α. Serum was run at equal volumes, while brain supernatant was run at 3.2 ug/uL in accordance with the instructions of the manufacturer’s protocol. In brief, the supernatant was incubated with antibody-coupled beads. After a series of washes, a biotinylated detection antibody was added to the beads, and the reaction mixture was detected by the addition of streptavidin-conjugated to phycoerythrin. The bead sets were analyzed using a flow-based Luminex™ 100 suspension array system on the Bio-Plex 200 platform . The unknown sample cytokine concentrations were then calculated by Bio-Plex Manager software using a standard curve derived from the known reference cytokine concentrations supplied by the manufacturer.The computational analysis was performed to predict metagenome functional content in each sample of the naïve, vancomycin, and ABX-cocktail groups. Briefly, the 16S ASVs were normalized and aligned to a reference phylogenetic tree then the predicted functional gene families and copy numbers for each specific ASV were found. We ran the full PICRUSt2 pipeline command in QIIME2. As output, we obtained MetaCyc pathway abundance predictions based on enzyme profile enzyme commission (EC) code and abundances . LEfSe ip < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The LDA threshold was set at ± 2. LEfSe data analysis was prepared using the Huttenhower Lab Galaxy server. Statistical analysis of the gut microbiota using two-way ANOVA followed by the Tukey post hoc test for multiple comparisons. This data is presented as mean ± standard error of the mean (SEM). LDA analysis incorporates both Kruskal–Wallis and pairwise Wilcoxon’s tests. The Kruskal–Wallis was used for the analysis of cytokine data, followed by Dunn’s post hoc test for multiple comparisons. The gut microbiome data was analyzed using Qiime2 software, and all data were graphed using GraphPad Prism v 9.0 Software . The final figures were assembled using Biorender software 2022 . With all significant differences measured represented by * It is known that events occurring during the perinatal period have severe consequences for long-term risk for disease or neurodevelopmental disorders like ASD and schizophrenia ,75,76,77Bacteroidetes or Actinobacteria or initiated as with Proteobacteria as compared to the naïve controls. Such shifts at the phyla level can be quite detrimental and have profound consequences for gut health.Therefore, this study addressed whether maternal antibiotic treatment during pregnancy evokes lasting changes in the gut microbiota and immunological responses of post-weaned prepubescent mice. We profiled the gut microbiota of the offspring and evaluated their serum and brain immunological cytokine levels in the early post-weaning period. To model this gut microbiota development in mice, we used prepubescent pups that stop drinking mother’s milk and switched to eating chow exclusively which begins at week 4. The weaning of mice comprises both dietary and environmental changes, which influence homeostasis and can lead to diet-induced dysbiosis . As showBacteroides spp. are completely missing from the ABX-cocktail group. The Bacteroides, has the capacity to produce an array of enzymes that breakdown carbohydrate [Parasutterella spp. are a vital component of the human and mouse gut microbiota [Lactobacillus spp. are wiped out in response to LPS. Lactobacillus spp. alter the expression of GABA receptors and influence the production of oxytocin [Enterobacteriaceae and Enterococcus spp. Enterobacteriaceae spp. are Gram-negative bacteria, some of which are considered commensal. However, some members are pathogenic and have been associated with inflammatory bowel disease [Enterococcus species typically are non-deleterious in healthy individuals; however, these can cause health problems in individuals with a weakened immune system [Though the shifts at the baseline phyla level did not reach significance, there are significant alterations at the genus level at baseline in response to maternal ABX-treatment. For instance, ohydrate , while Pcrobiota , is onlyoxytocin . Changes disease . The come system . When mie system ,92,93. Ie system ,94. In ge system , unlike e system ,97,98. TFactors from the circulation can ultimately reach the brain after crossing the blood–brain barrier (BBB), many of these could be derived from the gut and thus, there exists a form of cross communication with the GI system ,100. In The results of the current study also indicate that the more significant impairments in gut microbiome development were seen in 5-week-olds. Despite a number of factors changing at week 4, i.e., placed in a new cage (environment) and removed from milk (diet), we see greater alterations happening at week 5. In addition, the 5-week-old mice produce significantly more IL-13 and IL-17 than 4-week-old mice. Reduced proinflammatory cytokines are potentially due to reduced response to LPS challenge and may result in less immune and brain interactions. The extended dysbiotic period from infancy into the prepubescence stage, shown by decrease in bacterial diversity and decreased of key populations, may result in dysfunction in immune response . Recent Muribaculaceae family had many unclassified genera, so it remained categorized at the family level. FSuture studies will also include an assessment of cell-specific markers to identify the contribution of the immune cell to the altered immune response. Additionally, males and females will be analysed separately to determine the sex-specific contributions to the gut and immune profile in this prepubescent period, also whether changes in bacteria and brain cytokines could also lead to behavioral change, however, here we were focused on specific changes in gut microbiome and immune response at 4–5 weeks regardless of sex. Despite the aforementioned limitations, our findings suggest that the administration of antibiotics during pregnancy and delivery results in alterations to the gut microbiota and immune profile of offspring and can cause long-lasting changes. Our study has several limitations. First, we did not evaluate the maternal gut microbiota to confirm dysbiosis in the dams; we instead focused on the offspring. Based on published studies, the concentrations and timeframes we chose would elicit a dysbiotic response at the end of antibiotic treatment. These studies have demonstrated that maternal antibiotics during pregnancy would disrupt the maternal and offspring gut microbiota. We intentionally focused our study at 4 to 5 weeks and not across the life-span as little is known about this period of development. Additionally, components of the ABX-cocktail have the potential to cross the GVB. Therefore, we cannot rule out any direct impact on the embryos. Finally, limitations in sequencing prevent us from identifying the exact bacterial species for most reads, therefore, the genera was the lowest level of classification. Additionally, the In conclusion, maternal antibiotics during pregnancy results in modifications of gut microbiota in offspring mice in addition to the peripheral blood and the CNS immune response of offspring. These findings suggest that maternal antibiotics may be a risk factor for disrupted gut microbiota development in early life, resulting in long-term health deficits and leading to disease. This study highlights key gut microbiota and immune changes that occur during the post-weaning prepubescent stage can be altered by maternal ABX-treatments. Further investigation into the long-term effect of maternal ABX-treatment on offspring’s health should include more prepubescent mice.


T = 298.15 K and at atmospheric pressure (p = 0.1MPa) was measured with a drop volume tensiometer. These mixtures areformed by alkyl lactates (methyl lactate and ethyl lactate) and alcohols. Surface tension deviations for all these systems were calculatedand correlated with the mole fraction using the Myers–Scottequation. For the mixtures alkyl lactate with methanol, the surfacetension deviations are positive. With regard to the mixtures containingethanol, the surface tension deviation is positive for methyl lactateand slightly negative in the lactate-poor region for ethyl lactate.For the rest of the mixtures, the surface tension deviations are negative.A molecular interpretation of the different behaviors observed wasproposed.The surface behavior of 18 binary mixtures at  This interestin green chemistry urges the research community to introduce new approachesto industrial problems and take into account the biodegradabilityand environmental toxicology with renovated interest.3 The organicesters especially the lactates are between the chemicals studied.They can be used widely in the chemical industry applications inducingthe production of less contaminant solvents. In this context, we areinterested to give some approach of the thermophysical behavior ofbinary mixtures containing these green solvents and alcohols.The thermophysical behavior of different families of compoundsobtained from biomass and classified as green solvents has been studiedby our research group in the last years.The surface tension is considered one of the most important thermophysicalproperties, which have an interesting contribution to research ofthe liquid systems and to different industrial applications, for example,separations and extractions. The study of the surface tension allowsinterpreting some molecular interactions of liquids.n-alkanols from methanol to 1-hexanol and isomeric butanols)at T = 298.15 K and at atmospheric pressure. It canbe noted that we have previously studied bulk properties of some ofthese systems, methyl lactate and ethyl lactate with n-alkanols (from methanol to 1-butanol).4In thispaper, we present the surface behavior, trough surfacetensions, and surface tension deviations of the binary mixtures involvingalkyl lactates (methyl lactate and ethyl lactate) and alcohols , are summarized in For each binary mixture, the surface tensiondeviation was correlatedwith the molar fraction by means of the Myers–Scott equationn-alkanol (2) systems are presented. These surface tension deviationsshow three different behaviors. We can note that the methyl lactate+ methanol system shows positive deviations over the whole compositionrange, with a maximum found at around x1 = 0.5. However, Δσ values exhibit a sigmoid shape forthe methyl lactate + ethanol mixture. Being the surface tension deviationnegative in the lactate- poor region and becomes positive when x1 reaches 0.2. Finally, the mixtures methyllactate + n-alkanol (1-propanol to 1-hexanol) shownegative deviations. The minimum of the surface tension deviationoccurs in the lactate-rich region (0.65 < x1 < 0.7). Δσ values become more negative from1-propanol to 1-hexanol.In x1 < 0.75). Δσvalues are more negative in the following succeeding order: 2-methyl-2-propanol< 1-butanol < 2-butanol < 2-methyl-1-propanol.n-alkanol (2) are plotted. The surface tension deviations show twodifferent behaviors. Positive deviation is observed for the mixturesinvolving methanol and ethanol. The maxima occur at x1 = 0.5 and x1 = 0.6 for methanoland ethanol, respectively, while for the rest of n-alkanol (1-propanol to 1-hexanol), negative deviation is obtainedover the whole composition range. The minima occur at x1 = 0.65. The following order gives more negative surfacetension deviations: 1-propanol < 1-butanol < 1-pentanol <1-hexanol.In x1 <0.7) region. The following order gives more negative Δσvalues: 2-methyl-2-propanol < 1-butanol < 2-butanol < 2-methyl-1-propanol.Finally, n-alkanol chain.It can be noted that the influence of the butanol ramificationon the surface behavior is less marked than the length of the On the other hand, the binary mixturescontaining ethyl lactateshow bigger positive surface tension deviations or less negative surfacetension deviations than the systems with methyl lactate.17 With respect to structuralfactors, they are more related to the volumetric behavior.Thesurface tension is a thermophysical property related to bothmolecular interactions and structural factors between the mixed components.The surface tensions of alkyl lactates are bigger than the surfacetensions of the alkanols, showing that the cohesive interactions amongalkyl lactate molecules are stronger than those among the alkanolones. The positive surface tensions obtained for the systems containingmethanol and ethanol could be explained by strong interactions betweenalkyl lactates and methanol and ethanol. This heteroassociation maintainsthe methanol and ethanol molecules in the bulk, preventing their migrationfrom the bulk to the surface and leading to positive Δσvalues. For the rest of alkanols, the interactions between the componentsin the mixing process are lower than the weakening of self-interactionsin the pure liquids. Therefore, the compounds of lower surface tension,alkanols, experience surface migration, leading to negative surfacetension deviations.4 . The excess molar volumes depend on both the strengthof the molecular interactions and structural effects. For the systemsalkyl lactate with n-alkanol, n-alkanols,while for methanol and ethanol, some heteroassociation takes place,lowering the excess molar enthalpy. That is, something similar tothe comparison between volumetric and surface behavior occurs withcalorimetric and surface behavior.18Inthis sense, 4n-alkanols from methanol to 1-hexanol and isomeric butanols)is presented at T = 298.15 K and at p = 0.1 MPa.In this contribution, the surfacetension of binary mixtures involvingalkyl lactates (methyl lactate and ethyl lactate) and alcohols (Using a CP225-D Sartorius Semimicro mass balance,the mixtureswere prepared. Their surface tensions were determined by the dropvolume method using a Lauda TVT-2 tensiometer. For the calculationof the surface tension, the densities of the samples are needed, andthey were measured by means of an Anton Paar DMA-5000 densimeter.n-alkanols,Δσ becomes more negative with the length of n-alkanol, and for the isomeric butanols, the following order givesmore negative surface tension deviations: 2-methyl-2-propanol <1-butanol < 2-butanol < 2-methyl-1-propanol.The surface tension deviation for all the mixtures was calculated.For the mixtures alkyl lactate with methanol and ethanol, the surfacetension deviations are positive, although in the case of the ethyllactate + ethanol system, Δσ presents slightly negativevalues in the lactate-poor region. For the rest of the mixtures, thesurface tension deviations are negative. For


This study investigated the effects of a school-based skill-training intervention in attention control and executive functions for pupils with hyperactivity-impulsivity (H-I) and cognitive control (CC) deficits. The main aim was to examine whether the intervention differently influenced H-I and CC, and whether cognitive abilities or conduct problems moderated response to the intervention.n = 71) and a waitlist control group (n = 77). Intervention outcomes were assessed with an inventory assessing executive function difficulties (including H-I and CC) completed by classroom teachers.Elementary school pupils from 41 schools participated the study and were divided into an intervention group (Significant intervention effects and positive changes were detected in CC but not in H-I. Significant intervention effects were found mainly among pupils with low levels of conduct problems.The results suggest that a skill-training intervention has specific positive effects on CC, but conduct problems may diminish response to intervention. Executive functions (EF) enable us to control our thoughts and actions Barkley,  and are Studies focusing specifically on children with ADHD have found the three nominal subtypes of deficits  = 2.64, p = 0.172} or school {[F] = 1.44, p = 0.16} had no significant effect on the ATTEX gain score; thus, a single-level model could be used in the main analysis.Comparison between the intervention and waitlist group participants did not reveal statistically significant differences in age, grade level, ATTEX scores, conduct problem symptoms, WISC-IV scores, or mothers' education . The num2 = 41.19, p = 0.066). There were no statistically significant differences in the outcome or moderator variables as measured at T1 between pupils missing data from any timepoints compared to pupils with complete data except for age, where pupils with missing data were slightly older {[F] = 12.219, p < 0.001}.Missing data affecting pre- and post-assessments were mainly due to missing teacher ratings. Analysis of the randomness of missing of data using Little's CAR showed that it was completely at random ] = 14.97, p < 0.001, ηp2 = 0.14; pre- vs. follow-up [F] = 16.43, p < 0.001, ηp2 = 0.16}. The pupils in the low-fidelity intervention groups had, on average, more EF deficit symptoms in the post-assessments compared to the pre-intervention assessments  and even more symptoms in the follow-up than in the post-assessments . These six groups were excluded from the following analysis. In the second cohort social validity assessments were completed by 38 intervention providers . The results of the social validity assessments indicated high social validity, .Overall, 6 intervention groups (19 participants) out of 25 had lower than 80 % fidelity rate. Fidelity influenced the ATTEX Total gain score {pre- vs. post-test; [F] = 5.15, p = 0.025, ηp2 = 0.03; The analyses revealed that the intervention group improved more in CC compared to the waitlist group during the intervention {[F] = 3.42, p = 0.066} was found. The process moderator analysis showed that when the conduct problem score was low , there was a significant relationship between group and the intervention gain; Maltti participants had significantly higher gains in the CC compared to the waitlist participants if their conduct problem scores were low ] = 8.60, p = 004; ηp2 = 0.08}.A near statistically significant interaction effect of conduct problems and group on the ATTEX Total score and the CC {[were low . A totalThe main purpose of the study was to explore the effect of a school-based intervention  conducted as part of schools' special educational support on EF deficit symptoms observed in a classroom setting and, especially, to examine the moderators of the intervention response. The results revealed that the intervention supported the positive development of on-task skills and behaviors. More specifically, a significant intervention effect and positive change were detected in CC, but not in H-I, between pre- and post-assessments, which was in concordance with our expectations, as the intervention was targeted at skills that aim at improving attention and inhibition control in on-task situations and task completion strategies. Significant intervention effect was detected between pre- and post-assessments but not between pre- and follow-up assessments. Consistent with previous findings  did not moderate the response to the intervention, which is in accordance with a previous study by Langberg et al. . This suConduct problems did influence the intervention outcomes; moderator analysis showed that in the intervention group, improvements were demonstrated mainly among pupils with low levels (average or slightly raised) of conduct problems. Our results indicated that school-based training interventions that focus on on-task skills do not necessarily benefit pupils with higher levels (high or very high) of conduct problem symptoms, which is in line with Langberg et al. . There aPrevious research has shown that successful implementation of behavioral interventions and good implementation fidelity improve the intervention outcomes of evidence-based practices , Svenska Kulturfondet Stiftelsen (nos. 160026 for 2015-2019), Brita Maria Renluns Minne (nos. 20-1663 for 2015-2019), and Svenska Folkskolans Vänner (nos. SFV-0204 for 2015-2019).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aim of this study was to evaluate the association between preoperative IL-25 levels and HBV-HCC patient outcomes following liver surgery.via ELISA.This study enrolled consecutive HCC patients that had undergone liver surgery from 2008 to 2015. Baseline patient clinical properties were assessed to establish predictors of postoperative overall survival and recurrence-free survival  following liver resection. In addition, serum IL-25 levels were assessed p < 0.05). Cox multivariate regression analyses revealed an IL-25 level ≥ 14.9 μg/L to be an independent predictor of poorer RFS and OS. A combination of IL-25 levels and tumor diameter may be an even more reliable predictor of OS.Cox regression analyses revealed IL-25 levels to be independently related to the OS and RFS of 896 HBV-associated HCC patients. An optimal IL-25 cutoff level of 14.9 μg/ml was identified, with 206 patients in this cohort having IL-25 levels above this threshold. Both the OS and RFS of patients with an IL-25 level <14.9 μg/ml were significantly better after liver resection as compared to those of patients with higher preoperative levels of this cytokine (IL-25 levels are independent predictors of postoperative survival within HCC patients undergoing liver resection. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors of the digestive system, with high morbidity and mortality , 2. Its Therefore, it is very important to find new biomarkers related to liver cancer, achieve multi-indicator combined detection, improve the accuracy of early diagnosis of liver cancer, and reduce the rate of missed diagnosis. Over the years, other tumor markers for HCC have been proposed, such as Golgi protein 73 (GP73), Glypican-3 (GPC3), and cytokeratin 19 (CK-19) –8. GP73 Chronic inflammation is often a main driver of oncogenesis, and suppressing such inflammation can thus slow or arrest the physiological progression of cancer , 10. InfFor this study, HBV-infected patients that had undergone liver transplantation conducted by a single surgical team at the 900th Hospital of Logistics Support Force from January 2008 to June 2015 were retrospectively enrolled. All patients had been diagnosed with HCC as per the European Society for the Study of the Liver (EASL) criteria , with paFor the first 2 years after surgery, patients experienced follow-up every 3 months, and every 6 months thereafter. Hospital staff blinded to study objectives conducted all follow-up. All patients were regularly monitored for recurrence using approaches including AFP analyses, chest x-rays, and abdominal USG, MRI, or CT scans that were conducted every 3 months. HCC recurrence was diagnosed using the same criteria as were used to diagnose the primary disease before surgery. Approaches to treating recurrent diseases included TACE, PRFA, and PEI, with the exact procedure being selected based on patient- and tumor-specific factors.via a PSM approach as described previously  was performed. Specifically, cases with low and high IL-25 were matched eviously . Covariaeviously .Levels of serum IL-25 were measured in HCC patient samples with the Human IL-25 DuoSet ELISA kit (R&D Systems) based on the provided directions.t-tests or χ2 assessments as appropriate. The OS and RFS cases were assessed with Kaplan–Meier curves as well as log-rank measurements. Multivariate and univariate methods were used to guide the design of a prognostic nomogram, which was constructed with the “rms” package using R v.3.5.1 (http://www.r-project.org/). This nomogram was assessed based on measurements of the conformity index (C-index), with rcorrp.cens being used to compare the C-index values for this nomogram to those for other nomograms in Hmisc (p < 0.05 as the threshold of significance.All outcomes are given as median (range) or mean ± standard deviation (SD), and were studied by implementing unpaired Student’s in Hmisc . Analysen = 11), surgery (n = 5), liver failure-related mortality within 30 days postoperatively (n = 6), or clinically detected preoperative infection (n = 15), leaving a cohort of 896 patients eligible for these analyses , indicating that higher IL-25 levels are associated with more advanced HCC. After a PSM analysis, 156 patient pairs were generated (p > 0.05).Next, ROC curve analyses were used to establish an optimal IL-25 cutoff level capable of differentiating between HCC patient outcomes. The selected cutoff value was 14.9 μg/L, yielding an AUC value of 0.730, a specificity of 0.640, and a sensitivity of 0.757 Figure 2p < 0.05). Higher levels of IL-25 were also associated with decreased 3- and 5-year OS relative to lower IL-25 levels  . Similarly, following the PSM, the 3- and 5-year OS of cases with high IL-25 levels were 77.6% and 61.1%, respectively, which were significantly decreased as compared to those of cases with low IL-25 levels (p < 0.05) . The nomogram C-index value (0.726) for OS was higher than that for IL-25 (0.559), HBV-DNA (0.569), AFP (0.584), encapsulation of tumor (0.591), and diameter of tumor (0.635) . These results thus supported the predictive accuracy of these nomograms, with both the RFS and OS nomograms exhibiting AUC values higher than those for other prognostic risk factors  (p < 0.05), and in an analysis of multivariate for predictors associated with patient RFS, the HR for IL-25 was the greatest. As IL-25 exhibited the greatest specific weight of any factor in a predictive nomogram for HCC patient RFS, this suggested that IL-25 is the most robust predictor of RFS in this patient population  Table 8.The onset and progression of HCC are driven in large part by interactions between nascent tumor cells and the surrounding inflammatory milieu –22. Thervia a multivariate analysis approach, with elevated preoperative IL-25 levels being independent predictors of decreased OS and RFS in these cases. Importantly, high IL-25 levels functioned as an accurate predictor of long-lasting survival in cases with early-stage disease. While IL-25 levels were the best-identified predictor of RFS in this study, a combination of IL-25 levels and tumor diameter was better able to predict HBV-associated HCC patient OS. The mechanisms behind these effects are not fully understood, but some researchers believe that IL-25-induced dysregulation of intestinal microbiota promotes hepatocellular carcinoma through alternate activation of macrophages in the tumor microenvironment. Together, these results provide clear evidence that preoperative serum IL-25 levels can predict HCC patient prognosis.IL-25 is an inflammatory IL-17 family cytokine that is best studied as a driver of type 2 immune responses , 25–27. The primary limitation of this research is that it was a single-center retrospective analysis, and it is thus susceptible to potential bias with respect to patient selection. Future large-scale multi-center studies validating and expanding upon our results will thus be essential to affirm the clinical relevance of serum IL-25 as a prognostic biomarker in HBV-HCC patients.This study suggests that serum IL-25 levels may be an independent and useful tumor marker for the diagnosis of liver cancer. IL-25 is still valuable in the diagnosis of AFP-negative HCC and can be used as a supplement to AFP in the diagnosis of HCC. The combined diagnosis of the two markers greatly improves the early diagnostic accuracy of HCC. In addition, IL-25 values are associated with several pathological features that represent tumor aggressiveness and/or poor prognosis. Finally, IL-25 could help in the customized management of cases with risk factors for HCC recurrence after liver resection.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Ethics Committee of 900TH Hospital of Logistics Support Force (LLH-20150801). The patients/participants provided their written informed consent to participate in this study.Conception and design: S-hC and XW; Administrative support: S-hC and XW; Provision of study materials or patients: S-hC and XW; Collection and assembly of data: All authors; Data analysis and interpretation: All authors; Manuscript writing: All authors; Final approval of manuscript: All authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.